Tuberculosis (TB) is a devastating infectious disease caused by Mycobacterium tuberculosis that results in more than 8 million new cases and 1.4 million deaths every year. The efficacy of currently available anti-TB drugs has been compromised by the emergence of multidrug-resistant and extensively drug-resistant strains, therefore, there is an urgent need to develop novel therapeutic strategies against TB. In M. tuberculosis, three paralogous protein secretion systems - ESX-1, ESX-3 and ESX-5 - secrete PE-PPE dimers during defined stages of the infection process. PE and PPE proteins represent two large families of highly polymorphic proteins that are especially abundant in pathogenic mycobacteria. PE/PPE proteins are either cell wall associated or secreted into the extracellular milieu. These protein families are targets for vaccine development and diagnostic reagents. Multiple PE/PPE proteins play a role in in virulence, immune evasion and persistence in host. An outstanding question in the field is what the mechanisms of PE/PPE secretion and assembly in the cell wall of mycobacteria. This proposal is designed to help answer this important biological question by using a range of genetic, biochemical and structural approaches, including X-ray crystallography and mass-spectrometry. This study is significant because the protein secretion systems of M. tuberculosis, the ESX system in particular, are attractive targets for drug development. The results of our study will significantly advance our understanding of mycobacterial pathogenesis, and will provide structural knowledge that will guide the development of novel therapeutic targets and the design of a better vaccine against TB.